70
KONSTANTINOVA et al.
The reaction progress was monitored with HPLC: s, H%), 5.81 (1 H, d, J1',2' 3.7, H1'), 5.74 and 5.38
a detection wavelength for the control reaction was
254 nm, and for the test reactions was determined sepꢀ
arately for each modified base with a UV spectrophoꢀ
tometer (hence, the detection was conducted at two
wavelengths). A comparison of the hypoxanthine
amount (% according to HPLC data at 254 nm) was
conducted in the test and control mixtures.
(2 H, m, OH3' and OH2'), 4.89 (1 H, m, OH5'), 4.32
(1 H, m, H2'), 4.13 (1 H, m, H3'), 3.94 (1 H, m, H4'),
3.61 (1 H, dd, J4',5' 3.8, H5a'), 3.54 (2 H, m,
'
2C
'
2NH), 3.48 (1 H, m, J4',5' 4.89, H5'), 3.43
NCH
H
(2 H, m, NCH2CH2NH), 2.72 (2 H, m,
'
'
2NH), 2.65 (2 H, m, NCH2CH2NH).
NCH
2C
H
13C NMR: 160.55 (C(O)), 157.69 (C3), 144.83 (C5),
92.7 (C1'), 85.84 (C4'), 74.98 (C2'), 70.39 (C3'), 61.71
(C5'), 48.34 (NCH2CH2NH), 46.60 (NCH2CH2NH),
1ꢀβꢀDꢀRibofuranozylꢀ1,2,4ꢀtriazoleꢀ3ꢀcarbonitrile
(XXII). A (XVIII) base 20 mg (2 mmol), guanosine
451.97 mg (15 mmol), and KH2PO4 28.93 mg
(2 mmol) were dissolved in 106.3 mL of distilled water. 45.85 (NCH'2CH'2NH), 43.35 (NCH'2CH'2NH).
15N NMR: 282.33 (N of piperazinyl), 254.38 (N4),
The pH of the solution was adjusted to 7.0 with 2 mM
KOH. PNP (1.1 mL) was added to the reaction mixꢀ
ture. The solution was thermostated at 55°С for 4 d.
229.4 (N1), 122.44 (NH of piperazinyl).
The reaction mixture was placed in a refrigerator for
2 h for guanine precipitation. The precipitate was
removed with centrifugation (2 min, 12000 rpm), the
supernatant was separated, and the precipitate was disꢀ
persed in a minimal quantity of water followed by the
centrifugation. Supernatants were combined and conꢀ
centrated under vacuum (5 mmHg). A product was
isolated with preparative chromatography on the Luna
ACKNOWLEDGMENTS
The authors are grateful to R.S. Esipov and T.I. Muraꢀ
v’eva for providing preparations of purine nucleoside
phosphorylase. This work was supported by the Rusꢀ
sian Foundation for Basic Research, projects no. 07ꢀ
04ꢀ13573ꢀofiꢀts and 10ꢀ04ꢀ01020ꢀa, by the Federal
Targeted Program “National Technological Base” for
2007–2012 (state contract no. GP/07/439/NTB/K
from July 23, 2007), and by the Federal Targeted Proꢀ
gram “Scientific and ScientificꢀPedagogical Personꢀ
nel of Innovative Russia,” project no. P1085 from May
31, 2010.
C18 column (Phenomenx) 100 μm, 250 × 21.20 mm.
Fractions containing the product were combined, and
eluent was removed under vacuum (5 mm Hg). The
residue was dried in a vacuum desiccator. The yield
was 27 mg (60%), main compound content 98.35%,
Rt = 5.4 min (HPLC, variant 5), m/z: 227.0763 [M +
H]+ (calc. 227.0775). 1H NMR spectrum: 9.15 (1 H, s,
H5), 5.89 (1 H, d, J1',2' 3.7, H1’), 5.66 (1 H, br.s,
REFERENCES
OH2'), 5.24 (1 H, br.s, OH3', 4.93 (1 H, br.t,
J 4.5,
1. Hayden, F.G., Antiviral Res., 1996, no. 29, pp. 45–48.
OH5'), 4.34 (1 H, dd, 3.9, H'), 4.13 (1H, dd, J3',2' and
J
2. Galegov, G.A., L’vov, N.D., Petrova, I.G., and Floꢀ
rent’ev, V.L., Vopr. Med. Khim., 1986, vol. 32, no. 1,
pp. 10–19.
J3',4' 4.4, H3'), 3.98 (1 H, dd, J4',3' 4.4, J4',5' 9.1, H4’),
3.62 (1 H, m, H5a'), 3.51 (1 H, m, H5b'). 15N NMR:
307.7 (N2 triazole), 260.4 (N4 triazole), 237.2 (N1 triꢀ
azole).
3. Crotty, S., Maag, D., Arnold, J.J., Zhong, W., Lau, J.Y.,
Hong, Z., Andino, R., and Cameron, C.E., Nature
Medicine, 2000, vol. 6, pp. 1375–1379.
1ꢀβꢀDꢀRibofuranosylꢀ3ꢀcarboxpiperazinylꢀ1,2,4ꢀtriꢀ
azole (XXIII). KH2PO4 (0.034 g) was dissolved in
61.54 mL of distilled water. The pH of the buffer soluꢀ
tion was adjusted to 7.0 with 2 M KOH. Guanosine
(0.262 g, 15 mM and base (XVIh) (0.050 g, 3.75 mM)
were dissolved in the buffer. The reaction mixture was
4. Tam, R.C., Lau, J.Y., and Hong, Z., Antivir. Chem.
Chemother., 2001, vol. 12, pp. 261–272.
5. Cheshik, S.G., Vartanyan, R.V., Ivanova, L.A., Kisꢀ
teneva, L.B., and Galegov, G.A., Vopr. Virusol., 1992,
vol. 37, no. 2, pp. 97–99.
heated to 60°С to dissolve guanosine. Next, 0.06 mL
6. Bellobuono, A., Monadazzi, L., Tempini, S., Chiodo, F.,
of PNP was added to the reaction mixture. The soluꢀ
tion was themostated for 7 d. Guanine (according to
HPLC data) was filtered, and the filtrate was evapoꢀ
rated to a minimum volume. The residue was applied
to a column with reverseꢀphase sorbent Silica gel
100 С18ꢀreversed phase (Fluka) (size of the column
Magliano, E., Furione, L., and Ideo, G., J. Hepatol.
2000, vol. 33, pp. 463–468.
,
7. Lackner, H., Moser, A., Deutsch, J., Kessler, H.H.,
Benesch, M., Kerbl, R., Schwinger, W., Dornbusch, H.ꢀJ.,
Preisegger, K.ꢀH., and Urban, Ch., Pediatrics, 2000,
vol. 4, p. 106.
25
× 170 mm) and eluted with water. Fractions conꢀ
8. Hézode, C., Forestier, N., Dusheiko, G., Ferenci, P.,
Pol, S., Goeser, T., Bronowicki, J.ꢀP., Bourlière, M.,
Gharakhanian, S., Bengtsson, L., McNair, L., Kieffer, Sh.,
Kwong, A., Kauffman, R.S., Alam, J., Pawlotsky, J.ꢀM.,
and Zeuzem, S., N. Engl. J. Med., 2009, vol. 360,
pp. 1839–1850.
taining more than 95% of the target product were
combined, and the solvent was removed under vacuum
(5 mm Hg). The product was dried in a desiccator over
Р2О5 for 24 h. The yield was 47 mg (65%). The content
of the main compound was 98.28%, Rt = 2.22 min
(HPLC, variant 1);
314.1459), 336.1269 [
m
M
/
z
: 314.1452 [
M
+ H]+ (calc.
9. Kwong, A.D., Kauffman, R.S., Hurter, P., and Mueller, P.,
+ Na]+. 1Н NMR: 8.86 (1 H,
Nature Biotechnol., 2011, vol. 29, pp. 993–1003.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 39
No. 1
2013